Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-β-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72–96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation. Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America, and has a broad geographic distribution that runs from Mexico to Argentina [rgentina , 2.P. brasiliensis infection, the transition from mycelium to yeast cells represents an essential part of the overall virulence strategy of the fungus and is required for the establishment of PCM. The transition is stimulated by the rise in temperature that occurs when the inhaled mycelia or conidia contact the host lungs [During st lungs , 4.in vitro by shifting the incubation temperature from 26°C to 36°C (mycelia to yeast) or from 36°C to 26°C (yeast to mycelia) [This dimorphic fungal transition may be induced mycelia) . At 26°Cmycelia) . Using tmycelia) .Saccharomyces cerevisiae, chitin degradation and cell separation have been shown to depend on the action of a periplasmic acidic chitinase [Candida albicans is functionally homologous to the CTS1 gene of S. cerevisiae [The conversion from mycelia to yeast occurs in restricted regions of the mycelium , suggesthitinase . Additiorevisiae , where iP. brasiliensis yeast contains an N-acetyl-glucosamine- and chitin-binding lectin, named paracoccin (PCN), which also has N-acetyl-β-d-glucosaminidase (NAGase) activity [P. brasiliensis yeast cells were cultured in the presence of anti-paracoccin antibodies, fungal growth was reduced, yeast morphology was altered, and the chitin-labeling pattern at the cell wall was modified [We have previously shown that the cell wall of activity  and stimactivity . When P.modified . These oSince paracoccin binds to chitin and exerts chitinase activity, we hypothesized that paracoccin participates in the cell wall modification that occurs during the fungal transition from mycelia to yeast. To address this hypothesis, we took advantage of well characterized anti-paracoccin antibodies , 22 to ead libitum access to chow and water. Animal procedures were conducted in accordance with the ethical principles of animal research adopted by the Brazilian Society of Laboratory Animal and approved by the Ethical Committee for Ethics in Animal Research (CETEA) of the Ribeirão Preto School of Medicine, University of São Paulo, under protocol number 145/2005.BALB/c mice of 6–8 weeks of age were maintained in the animal facility of the Molecular and Cellular Biology Department, Ribeirão Preto School of Medicine, University of São Paulo. Animals were housed in individually ventilated cages, in light-tight cabinets, under a 12 h light-dark cycle and maintained at 20–22°C, under optimized hygienic conditions, and with P. brasiliensis isolate Pb18 was used in this study. Yeast cultures were grown in YPD medium (1% yeast extract (w/v), 2% peptone (w/v), 2% glucose (w/v), pH 6.5), at 37°C on an orbital shaker and subcultured every 7 days. The fungal mycelium phase (M) was obtained according to the method of [HIII mice, which are high antibody producers , were usP. brasiliensis cells were processed as previously described [For DIC microscopy, escribed .g. The pellets were embedded in OCT130 Tissue-Tek (Electron Microscopy Sciences), and the samples were frozen in acetone cooled with dry ice. After freezing the samples were stored at -20°C. Six-micron sections of the frozen samples were obtained using a cryostat , and placed on gelatinized glass slides. Before immunostaining, the slides were rinsed three times with PBS and blocked with 3% BSA in PBS for one hour at RT. The samples were incubated with mouse polyclonal anti-paracoccin IgG (150 μg/mL) or with anti-paracoccin IgY (1:100), as specified in the figures legends, diluted in blocking buffer for 2 h, at RT. The samples were rinsed with PBS and incubated for 1 hour in the dark with secondary antibodies: goat anti-mouse IgG conjugated with Alexa 488 or donkey IgG anti-chicken IgY conjugated with Alexa 488, diluted 1:1000 in blocking buffer. For detection of chitin on the cell wall, the samples were incubated with 10 μg/mL of wheat germ agglutinin conjugated to Texas Red®-X  in PBS for 1 h at RT in the dark. For detection of the fungal nucleus, samples were incubated with 2.5 μg/mL of DAPI . After ten washes with PBS for 5 minutes each, the slides were rinsed in ultrapure water and coverslips mounted with Fluoromount-G . The samples were examined with a TCS SP5 scanning confocal microscope . Serum from pre-immune HIII (1/250) mice or pre-immune IgY (obtained from chicken eggs before immunization) and secondary antibodies alone were used as controls  from mycelium-to-yeast and yeast-to-mycelium. The total RNA from the P. brasiliensis morphological forms was obtained using Trizol . Briefly, cells were harvested, disrupted by grinding in liquid nitrogen, and mixed with Trizol, according to the manufacturer’s instructions. Total RNA was treated with DNase I (Thermo Fisher Scientific Inc.). cDNA was synthesized using 1 μg of total RNA with oligo-dT12-18 primer (Thermo Fisher Scientific Inc.) and SuperScript III reverse transcriptase (Thermo Fisher Scientific Inc.), according to the manufacturer’s instructions. Real-time PCR was performed using Kit Platinum SYBR Green qPCR SuperMix-UDG with ROX (Thermo Fisher Scientific Inc.), according to the manufacturer’s instructions. The following specific primers were used for PCN-RT: 5´-CCGCCCCTTTGGTGATGT-3´ (sense) and 5´-TCGAAAAGCTCTCCCACTTC-3´ (antisense). To measure the gene expression levels, we used CFX96 real-time PCR detection system . Fold changes in mRNA expression were calculated using the 2-ΔCq formula, where ΔCq is the difference in the threshold cycle (Cq) between the target gene and the β-actin (Sequence ID: ref |XM_010763641.1|) and α-tubulin references genes [5′-GGATGAGGAGATGGATTATGG-3′ (sense) and 5′-GAAACACTCGACGCACACGAC-3′ (antisense); and 5′- GTGGACCAGGTGATCGATGT-3′ (sense) and 5′-ACCCTGGAGGCAGTCACA-3′ (antisense), respectively.The gene expression profile of es genes . The reaP. brasiliensis hyphae was carried out by confocal microscopy, using specific anti-paracoccin antibodies  Figs  and 4. TP. brasiliensis, we evaluated if there were differences in the relative expression of PCN mRNA in yeast, hypha, and transition forms. We compared the normalized mRNA-expression levels detected in the different fungal forms. PCN transcripts were detected in all evaluated morphological forms. Higher expression was verified in hypha and yeast-to-hypha transition forms in comparison to the expression in yeast and hypha-to-yeast transition forms , which also has N-acetyl-β-d-glucosaminidase (NAGase) activity [P. brasiliensis yeast cells were cultured in the presence of anti-paracoccin antibodies, fungal growth was reduced, yeast morphology was altered, and the chitin-labeling pattern at the cell wall was modified [We have previously shown that the cell wall of activity  and stimactivity . When P.modified . These oFungal adaptation to the host environment is associated with changes in the cell wall, which include structural alterations in carbohydrate polymers . As the P. brasiliensis because, depending on their distribution, molecules expressed during this period may play distinct roles in the fungal transformation process and influence the pathogenesis of paracoccidiodomycosis [Candida albicans hyphae and P. brasiliensis yeasts [P. brasiliensis transition from mycelial to yeast form. Indeed, we have shown recently that silencing the gene encoding paracoccin results in the development of yeasts that are deficient in cell segregation and are unable to do the transition from yeast to mycelia [We investigated the mycelium-to-yeast transition forms of omycosis . For exaomycosis , 30. Higs yeasts , 32. Our mycelia .P. brasiliensis following temperature shift. The features we observed were consistent with both original reports [We validated the model used in this study by kinetically analyzing the morphological features of  reports , 28, 34  reports –38 of th reports . The tra reports , 15. We  reports .Candida albicans, whose vesicles contain enzymes implicated in the biosynthesis of the new cell wall [P. brasiliensis hyphae as well defined as the Spitzenkörper in C. albicans.All the examined fungal forms displayed paracoccin, as detected by immunofluorescence staining. Inside mycelia, the fine punctate labeling was more concentrated near the tip; this paracoccin labeling was partially coincident with chitin detection, a finding consistent with our previous demonstration that paracoccin interacts with chitin , 21, 24.ell wall , 41. In ell wall , 42, 43.P. brasiliensis from hyphae to yeast, cracking of the outer electron-dense layer of the cell wall facilitates the formation of the yeast cell wall from the inner layer of the hyphal cell wall [P. brasiliensis dimorphism demonstrated that modifications in cell wall glucans play a fundamental role in fungal differentiation and morphogenesis [P. brasiliensis mycelium-to-yeast transition, instead of being evenly distributed throughout the morphotypes, were distinctly prominent in the areas of fungal growth. Consistent with this observation, paracoccin was extremely concentrated in the growth regions of all P. brasiliensis morphotypes. In mycelia, paracoccin was concentrated in the fungal tips and lateral protrusions. This type of organization is considered essential for the delivery of growth factors and enzymes to the specific regions of the hyphae, contributing not only to the maintenance of cell wall integrity but also to chitin degradation and hyphal prolongation. In transition forms, paracoccin was detected in lateral swellings of differentiating hyphae from which chlamydoconidia arise. In yeasts, the presence of paracoccin in budding regions is also compatible with a role in cell growth. Corroborating with these results of PCN localization during the morphological transition process, we verified the PCN mRNA expression in all P. brasiliensis morphotypes and during the transition phases from hypha-to-yeast and yeast-to-hypha. It is worth mentioning the higher PCN transcript expression in hyphae, which is the fungal infecting phase, and during yeast-to-hypha transition. These results are coherent with the proposed direct involvement of PCN in the morphological transition process, and consequently, in the cell wall remodelling, as previously suggested [It is known that during the transformation of ell wall , 34. Dirogenesis . Enzymesuggested .P. brasiliensis pathogenesis.In conclusion, this work provides support for the postulation that paracoccin plays a role in fungal growth and morphogenesis, participates in dimorphic fungal transformation, and, consequently, contributes to S1 FigP. brasiliensis hyphae (A–C) and yeasts (D–F) were incubated with chicken pre-immune IgY (A and D) and / or with the secondary antibody (B and E). The images show that there was no unspecific reaction. DIC microscopy of the stained samples (C and F). Similar results were obtained when used serum from pre-immune HIII mice (not shown).We considered that to give a better idea of the PCN localization it was important to show the DIC images of the morphotypes from which the data was derived. (TIF)Click here for additional data file.S2 FigMycelia cultured in liquid medium were induced to undergo yeast transformation by shifting the temperature from 26°C to 37°C. Samples were harvested at 12 h (A–C), 24 h (D–F), and 48 h (G–I) after the temperature shift and immunostained for paracoccin (PCN), using chicken IgY anti-paracoccin antibody conjugated to Alexa Fluor 488 (green) and stained with WGA (red) for chitin, and with DAPI (blue) for DNA.(TIF)Click here for additional data file.S3 FigMycelia cultured in liquid medium were induced to undergo yeast transformation by shifting the temperature from 26°C to 37°C. Samples were harvested at 72 h (A–C), 96 h (D–E), and 120 h (F–G) after the temperature shift and immunostained for paracoccin (PCN), using chicken IgY anti-paracoccin antibody conjugated to Alexa Fluor 488 (green) and stained with WGA (red) for chitin, and with DAPI (blue) for DNA.(TIF)Click here for additional data file.S4 FigP. brasiliensis yeast cells were stained for detection of paracoccin (A and C), using chicken IgY anti-paracoccin antibody conjugated to Alexa Fluor 488 (green), and for chitin (B and D) with Texas Red®-X WGA (red).(TIF)Click here for additional data file.


P = 5.80E-10 in European population. In Chinese population, several genetic association studies have investigated the association between rs3802842 variant and CRC risk. However these studies reported both positive and negative association results. It is still necessary to evaluate a specific variant in a specific population, which would be informative to reveal the disease mechanism. Until recently, there is no a systemic study to evaluate the potential association between rs3802842 and CRC risk in Chinese population by a meta-analysis method. Here, we aim to evaluate this association in Chinese population by a meta-analysis method using 12077 samples including 5816 CRC cases and 6261 controls. We identified the T allele of rs3802842 to be significantly related with an increase CRC risk  in Chinese population.A genome-wide association study identified a common genetic variant rs3802842 at 11q23 to be associated with CRC risk with OR=1.1 and  P = 5.80E-10 in European population . In CC vs. CA+AA model, we applied the fixed-effect model to perform the meta-analysis, which indicated significant association between rs3802842 CC genotype and CRC risk with P=2.22E-07, OR=1.39, and 95% CI . In CC+CA vs. AA model, we applied the random-effect model to perform the meta-analysis, which indicated significant association between rs3802842 CC+CA genotype and CRC risk with P=9.00E-03, OR=1.37, and 95% CI . The detailed information is described in Figure In C vs. A model, we applied the random-effect model to perform the meta-analysis, which indicated significant association between rs3802842 C allele and CRC risk with P=0.81 for C vs. A model.The possible publication bias of meta-analysis is evaluated by both funnel plot and a regression based statistical approach. Based on the shapes of funnel plots, we did not observe any asymmetric signal in all these three models as described in Figure A leave-one-out sensitivity analysis showed that the pooled ORs were not significantly changed when all these studies were excluded one by one, which indicated that the meta-analysis results were robust and reliable (data not shown).2 = 4.21, df = 3 (P = 0.24); I2 = 29%. We applied the fixed-effect model to perform the meta-analysis, which indicated significant association between rs3802842 C allele and CRC risk with P=9.19E-15, OR=1.31, and 95% CI . In the combined Hong Kong Chinese and Taiwan Chinese subgroup, we did not identified significant heterogeneity in these four studies with Heterogeneity: Chi2 = 0.00, df = 1 (P = 0.98); I2 = 0%. We applied the fixed-effect model to perform the meta-analysis, which indicated no significant association between rs3802842 C allele and CRC risk with P=0.08, OR=1.08, and 95% CI .In Han Chinese subgroup, we did not identified significant heterogeneity in these four studies with ChiTenesa et al. identified rs3802842 to be significantly associated with CRC risk . InteresUntil recently, six independent case-control association studies have been conducted to investigate the association between rs3802842 and CRC risk in Chinese population. Three studies reported positive association results –20, and In our study, we identified significant heterogeneity in these six genetic association studies. We think this may be caused by the substantial genetic variation in Han Chinese population . Chen et2 = 29%) is higher compared with that in combined Hong Kong and Singapore Chinese subgroup (I2 = 0%). Meta-analysis further showed the rs3802842 variant to be significantly associated with CRC risk in Han Chinese subgroup, but not in the combined Hong Kong and Taiwan Chinese subgroup.Considering the significant heterogeneity, we further performed a subgroup analysis in the Han Chinese subgroup, and the combined Hong Kong and Taiwan Chinese subgroup. The results are consistent with previous findings. The heterogeneity in Han Chinese subgroup  using the key words ‘rs3802842’ + ‘colorectal cancer’ . We also manually examined additional studies from the references cited in the original literature using Google Scholar database (https://scholar.google.com/), especially all associated publications citing the original CRC GWAS [Two reviewers independently selected the potential studies by systematically searching the PubMed database  be a case-control design in Chinese population; (2) evaluate the association between rs3802842 and CRC risk; (3) provide the original genotype number, or allele number, or odds ratio (OR) with 95% confidence interval (CI) for one of the three genetic models; or (4) provide sufficient data to calculate the genotype number, or allele number, or OR and 95% CI for one of these three genetic models. We excluded those studies that did not meet the inclusion criteria in following meta-analysis.We extracted (1) the name of the first author; (2) the year of publication; (3) the population; (4) the numbers of CRC cases and controls. Two reviewers independently extracted the data carefully. Meanwhile, a third reviewer resolved any disagreement. More detailed information has been widely described in previous studies using the meta-analysis methods –44.In brief, we used Review Manager 5.1 to investigate the potential heterogeneity in all the selected studies by a Cochran's Q test, calculate the pooled OR by a fixed effect model or a random-effect model based on the potential heterogeneity, determine the significance of pooled OR by a Z test.We calculated the Hardy-Weinberg equilibrium by a chi-square test in R program, if one study provides the control genotype number , 46. If We investigate potential publication bias by a funnel plot based approach, and a regression based statistical approach proposed by Egger. We performed a sensitivity analysis by a leave-one-out method . We evalWe performed a subgroup analysis in the Han Chinese subgroup including four studies, and in the combined Hong Kong Chinese and Taiwan Chinese subgroup including two studies using C vs. A model.


Phyllomedusa hypochondrialis through cloning of its precursor-encoding cDNA from a skin secretion-derived cDNA library by a 3′-rapid amplification of cDNA ends (RACE) strategy. Subsequently, the mature peptide was isolated and characterized using reverse-phase HPLC and MS/MS fragmentation sequencing. DLP-PH adopted an α-helical conformation in membrane mimetic solution and demonstrated unique structural features with two distinct domains that differed markedly in their physiochemical properties. Chemically synthesized replicates of DLP-PH showed antimicrobial activity against planktonic bacterial and yeast cells, but more potent against Escherichia coli at 32 μg/mL. However, DLP-PH showed much weaker inhibitory activity against the growth of sessile cells in biofilms. In addition, DLP-PH exhibited anti-proliferative activity against human cancer cell lines, H157, and PC3, but with no major toxicity against normal human cell, HMEC-1. These combined properties make DLP-PH deserving further study as an antimicrobial agent and further investigations of its structure-activity relationship could provide valuable new insights into drug lead candidates for antimicrobial and/or anti-cancer purposes.Amphibian skin secretions are an important treasure house of bioactive antimicrobial peptides (AMPs). Despite having been the focus of decades of research in this context, investigations of phyllomedusine frogs continue to identify new AMPs from their skin secretions. In this study, the prototype of a novel family of AMP distinctin-like-peptide-PH (DLP-PH) was identified from the skin secretion of the otherwise well-studied Tiger-Legged Tree Frog  Not even a century after the discovery of penicillin marked a new era is the treatment of bacterial infectious agents, modern medicine is quickly reaching a crossroads with respect to the utility of antibiotics in health care. Two trends are especially apparent and worrisome in what is an evolutionary arms race. The first is the recent, rapid increase in the emergence of drug-resistant infectious agents (“superbugs”) caused, in part, through the inappropriate prescribing of conventional antibiotics Poole, . The secIn this second context, antimicrobial peptides (AMPs) have attracted the attention of many researchers because of their numerous unique advantages, such as potent effects against a variety of pathogens, including many superbugs, their ability to suppress biofilm formation  coming from amphibians and 90% of those from frogs and toads. Nevertheless, the potential of amphibian skin secretions as a source of AMPs and of the AMPs themselves as a source of novel antibiotics has barely been tapped. Many new amphibian AMPs remain to be discovered and the AMPs that are already known either possess potent antimicrobial activities that deserve further study or can be used as templates for computer-aided drug design to enhance their efficacy.An important natural resource for AMPs, are the skin secretions of amphibians, which, in addition to AMPs, also contain a cocktail of other chemically complex bioactive molecules displaying a wide range of activities. Indeed, amphibians AMPs have attracted much attention over the past decades , displayed a number of interesting characteristics deserving further investigation, including an evaluation of its bioactivities.Within this latter context, we describe a novel AMP precursor that we identified from the skin secretion of P. hypochondrialis were obtained from a commercial source in Peru . The frogs were housed in a tropical frog vivarium at 25°C and 85% humidity under a 12 h/12 h day/night cycle and were fed multivitamin-loaded crickets three times per week. The skin secretions were sampled via the minimally invasive method of mild transdermal electrical stimulation . The polyadenylated mRNA was isolated by using the magnetic oligo-dT beads in accordance with the manufacturer's instructions. The isolated mRNAs were then reverse-transcribed to construct a cDNA library using a SMART-RACE Kit  and the 3'-RACE reactions to acquire full-length prepropeptide nucleic acid sequence data. Potential AMPs were then amplified from this library through the combination of the nested universal primer (5′-AAGCAGTGGTATCAACGCAGAGT−3′) that was supplied with the kit with a degenerate primer (5′-ACTTTCYGAWTTRYAAGMCCAAABATG-3′) that we designed to bind to a highly-conserved segment of the 5′-untranslated region of bioactive peptide cDNAs from phyllomedusine frogs  trifluoroacetic acid (TFA)/water and centrifuged for clarification. The supernatant was then analyzed using a gradient RP-HPLC system  fitted with an analytical column . Contents were eluted from the column using a linear gradient composed of 0.05/99.95 (v/v) TFA/water to 0.05/19.95/80.00 (v/v/v) TFA/water/acetonitrile over 240 min at a flow rate of 1 mL/min. The effluents were collected at 1-min intervals and their UV absorbance were monitored at both 214 and 280 nm simultaneously. Samples from each fraction were subsequently analyzed by MALDI-TOF MS  using α-cyano-4-hydroxycinnamic acid as the matrix. The fraction that contained a peptide with a molecular mass coincident with the mass of the mature peptide deduced from the cloned precursor-encoding cDNA was infused onto an LCQ-Fleet ion-trap MS  for fragmentation sequencing.Another five milligrams of the lyophiled skin secretion of Following the unequivocal confirmation of the primary structure of the mature peptide in the preceding step, the native peptide as well as three truncated versions of it  were chemically synthesized via solid-phase Fmoc chemistry using a Tribute automated synthesizer . Corresponding Wang-resins, protected amino acids, N-methylmorpholine (NMM), piperidine, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N,N-dimethylformamide (DMF), and dichloromethane (DCM) were used during the synthesis. The TFA/triisopropylsilane (TIPS)/water cocktail was used to cleave the peptide from the resin and to deprotect the side-chains. All peptides were analyzed and purified using RP-HPLC. Both molecular masses from MALDI-TOF and MS/MS fragmentation profiles were also employed to determine the purity and authenticity of the respective structures.In the first instance, secondary structures of the synthesized peptides were determined via circular dichroism (CD) using a JASCO J-815 CD spectrometer . The peptides were dissolved initially in 10 mM ammonium acetate and 50% TFE in 10 mM ammonium acetate to reach a concentration of 100 μM and then added into a 1-mm high-precision quartz cell . CD spectra were obtained at 20°C from 190 to 250 nm at a scanning speed of 100 nm/min with 1 nm bandwidth and 0.5 nm data pitch.http://rzlab.ucr.edu/scripts/wheel/wheel.cgi) was used to visualize the α-helical peptide and the I-TASSER online server .As DLP-PH was more effective against the growth of Staphylococcus aureus (NCTC 10788), the gram-negative bacteria Escherichia coli (NCTC 10418) and Pseudomonas aeruginosa (ATCC 27853), and the yeast Candida albicans (NCYC 1467) using the micro broth dilution method  was also used to determine the MICs in the presence of cations. For all assays, MICs were defined as the lowest concentration of peptides that resulted in the absorbance less than 0.1 in the 96-well plates whereas MBCs were defined as the lowest concentration that showed no colony growth on MHA plates.The antimicrobial activities of the synthetic peptides were assayed against both planktonic microbial cells as well as sessile cells in biofilms. In the former case, we quantified both the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) against planktonic cells of the gram-positive bacterium C. albicans was cultured in RPMI-1640 and the other strains were cultured in MHB with 2% glucose, respectively. Overnight cultures were washed once with pre-warmed PBS and diluted with fresh medium to 107 CFU/mL. For the MBIC assay, the peptide stock solutions were prepared in the same way with the same concentration range as for the MIC assays and then incubated at 37°C for 24 h. Thereafter, the plates were washed twice with PBS followed by the addition of 200 μL fresh MHB with 2% glucose  and 50 μl 1% TTC (g/v) solution per well. After 5 h incubation, 200 μL of the supernatant from each well were transferred to a new plate and its absorbance at 470 nm was determined using a Synergy HT plate reader . For the MBEC assay, 200 μL of the same diluted inoculum was dispersed into each well in a flat-bottomed 96-well plate for 48 h to form mature biofilms. Following sufficient time, the plates were washed three times with PBS to remove the planktonic cells and incubated with the same concentration range of peptide solutions as above at 37°C for 24 h. Thereafter, each well was washed, refilled with fresh medium, stained with TTC, and incubated for a further 5 h. Absorbance was measured in the same way as with the MBIC assays. MBICs and MBECs were determined as the lowest concentrations at which no colourimetric metabolites were formed by the bacteria.Susceptibility assays against sessile cells in biofilms were conducted by quantifying the minimal biofilm inhibition (MBIC) and the minimal biofilm eradication concentrations (MBEC) as well as the biofilm initial attachment inhibition against all the strains. MBIC and MBEC testing was performed, with minor modifications, according to Knezevic and Petrovic  and SabaP. aeruginosa, a strong biofilm-producing bacterium, using a slightly modified version of the method of Zhang et al.  inhibition assay was performed using g et al. . BrieflyS. aureus, E. coli, and C. albicans were washed with pre-warmed PBS and diluted to 5 × 105 CFU/mL in PBS. The synthesized peptides to be tested were dissolved in PBS and 2-fold diluted as described above to achieve a final concentration range from 512 to 1 μg/mL. Triton-X 100 (0.2%) in PBS was used as a positive control, with 100 μL of different concentrations of the peptide in PBS (512 to 1 μg/mL) being used as blank controls. The plates were incubated at 37°C for 2 h before the contents in each well were filtered through 0.22-μm syringe filters  into new plates. A volume of 100 μL of the supernatant was transferred into a new 96-well plate and the absorbance at 260 nm was measured using a plate reader.As an indicator of the potential lysis of the microbial cell membranes in the presence of the peptides, we performed a cytoplasmic material leakage assay  in physiological PBS. Each peptide was tested within the concentration range of 1 to 512 μg/mL (see above), with each peptide concentration being incubated with the red blood cell suspension at 37°C for 2 h. PBS solution and an equal volume of PBS solution containing 2% of Triton X-100 , a non-ionic detergent, were used as negative and positive controls, respectively. After 2 h, each peptide-cell mixture was centrifuged and 200 μL of supernatant were added to each well of a 96-well microtiter plate. The degree of lysis of the red blood cells was assessed by measuring the absorbance at 550 nm using a microplate reader.Possible anti-cancer activities of the novel peptide were investigated against a range of five human cancer cells lines: human prostate carcinoma cell line PC-3 (ATCC-CRL-1435) and human non-small cell lung cancer cell line H-157 (ATCC-CRL-5802) were cultured in RPMI-1640 medium  with 10% fetal bovine serum (FBS)  and 1% Penicillin-Streptomycin (PS) , whereas human breast melanoma cell line MDA-MB-435s (ATCC-HTB-129), human breast adenocarcinoma cell line MCF-7 (ATCC-HTB-22), and human neuronal glioblastoma cell line U251MG (ECACC-09063001) were cultured in DMEM medium  with 10% FBS and 1% PS. As a control, human microvessel endothelial cell line HMEC-1 (ATCC-CRL-3243), which was cultured in 10% FBS, 10 ng/mL epidermal growth factor (EGF) , 10 mM L-glutamine  and 1% PS loaded MCDB131 medium , was used to evaluate the cytotoxicity of the synthetic peptides on normal human cell line.−3 μg/mL. After the incubation period, 10 μL of 5 mg/mL MTT solution  were added to each well and incubated for a further 4 h. All cells were cultured in an incubator with a humidified environment with 5% CO2 at 37°C. Finally, the supernatant in each well was removed and replaced with 100 μL dimethylsulfoxide (DMSO) and the absorbance of each well at 570 nm being determined using a plate reader.Cell antiproliferative activity was assessed using the MTT cell viability assay. Briefly, the cells were counted and seeded in 96-well plates at a density of 5000 cells per well for 24 h. Thereafter, the cells were synchronized by treating them with serum-free medium for a further 12 h after which they were incubated for 24 h with the peptide dissolved in serum-free medium over a concentration range of 419.7 to 4.197 × 102O were applied as positive and negative control, respectively. After incubation, 50 μL of each sample medium were transferred into another 96-well plate and mixed with 50 μL of LDH reaction mixture. Each sample was further mixed with 50 μL of stop solution after a 30 min incubation period with the absence of light at room temperature. The LDH activity was measured by the absorbance at 490 nm, which subtracted the absorbance at 680 nm.Cell cytotoxicity activity was assessed using the LDH assay , and the experiment followed the instruction provided with the kit. The cells were seeded in a 96-well plate as described above and synchronized for a further 4 h. Afterwards, the cells were treated with the same concentration range of DLP-PH as used in the MTT assay and incubated for 45 min. Lysis solution and ddHt-test and one-way ANOVAs, with the statistical significance of the differences being indicated as *(p < 0.05), **(p < 0.01), and ***(p < 0.001). The dose-response curves were constructed using a “best-fit” algorithm and HC50 and IC50 values were calculated through the data-analysis package in GraphPad Prism 6 .For all assays, all peptide concentrations and controls were tested in three independent experiments of five replicates each. The statistical analyses employed P. hypochondrialis skin secretion derived cDNA library, we consistently retrieved a cDNA encoding the precursor of a novel peptide (DLP-PH) with an open reading frame of 80 amino-acid residues, including a signal peptide terminating in a cysteine residue; an acidic amino-acid residue-rich spacer peptide; and a predicted putative mature peptide of 36 amino-acid residues. The predicted mature peptide appeared after a lysine-arginine (KR) motif, which is a typical propeptide convertase cleavage processing site . Additionally, the DLP-PH peptide precursor demonstrated strong similarity to the B-chain peptide (distinctin-B) precursor of the heterodimeric AMP distinctin from Phyllomedusa distincta  of DLP-PH were predicted to face the cell membrane through electrostatic attraction  while avoiding Lys14, which might be enzymatically sensitive. Compared with DLP-PHc, DLP-PHt displayed a higher hydrophobicity (H = −0.283) and amphipathicity (μH = 0.270), but with values that were still distinctly lower than those for DLP-PH and DLP-PHn.The best molecular docking model between DLP-PH and the model of the n Figure . Both thn Figure  indicaten Figure  and C-ten Figure . Thus, wn Figure . A thirdS. aureus or in the presence of divalent cations  is nearly half of that of DLP-PH (4197 Da), their MICs against S. aureus were quite similar when viewed in terms of molarity: 62.2 vs. 61.0 μM, respectively]. The efficacy of DLP-PH against sessile cells of all strains in biofilm was strongly reduced compared to the analogous values against planktonic cells   Figure .S. aureus (256 μg/mL), barely any cells of E. coli were lysed at its MIC of 32 μg/mL. By contrast, DLP-PH continued to result in >50% cell lysis of C. albicans even at concentrations well-below its MIC (64 μg/mL) against this organism.The cytoplasmic materials release experiment revealed that DLP-PH lysed nearly 100% of the microbial cells at high concentrations of peptide  was only shown against the two cell lines H-157 and PC-3, with ICy Figure . By conte Figure . The LDHe Figure .Phyllomedusa hypochondrialis represents a well-studied frog species with respect to its skin secretions, with the Antimicrobial Peptide Database listing at least 36 AMPs that have been isolated from this species. Our study identified another novel AMP for the species, with the mature peptide, DLP-PH, First, as the name suggests, showing high identity to the 25-residue B-chain of the AMP distinctin from Phyllomedusa distincta  for this organism, implying some other mechanism for the observed bioactivity . Similarly, the high leakage of cytoplasmic contents that was inferred for C. albicans below its observed MIC (64 μg/mL), indicates that cell lysis alone appears to be insufficient for antimicrobial action and that an additional, possibly intracellular, mechanism is also required. Moreover, the antimicrobial potency of DLP-PH was extremely decreased by the biofilm-forming sessile cells, which indicates it failed to inhibit or eradicate microorganism biofilm, whilst DLP-PH actually inhibited the initial attachment of P. aeruginosa at a lower concentration than its MIC. Apparently, such interaction is not able to induce either the lethal of bacterial killing or inhibiting the biofilm formation, we therefore assume that DLP-PH may further interfere the attachment in a non-lethal mechanism, which was reported that cationic AMPs may bind to bacteria cell or cover the non-living surface to reduce bacterial adhesion  of antimicrobial action of AMPs in general, thereby helping to direct drug design . The anti-biofilm activity of DLP-PH, although weaker than its general antimicrobial activity, is also worthy of further study as a potential new therapeutic direction in the fight against chronic diseases caused by drug-resistant biofilms. Finally, the fact that we discovered DLP-PH, which not only represents a novel AMP, but might also be a prototype peptide of an entirely new AMP family, in an otherwise well-studied frog species argues for the continued investigation of the skin secretions of amphibians to potentially provide many more new leads for the therapeutic application for antibiotic design and development.The conception and design of study was conducted by LW, MZ, and TC. The laboratory work and acquisition of data was performed by DW, YG, YT, and YL. The analysis of data was conducted by CM and XX. The manuscript was drafted by YG, DW, YT, and CM, and revised by OB-E, XX, and CS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Liver disease is a frequent cause of morbidity and mortality in HIV infection. We examined the relationship of cocaine use, liver disease progression and mortality in an HIV-infected cohort.Consent was obtained from 487 HIV+ participants, a subset of the Miami Adult Studies on HIV (MASH) cohort. Participants were eligible if they were followed for at least two years, completed questionnaires on demographics and illicit drug use and had complete metabolic panels, CD4 cell counts and HIV-viral loads. FIB-4 was calculated and cut-off points were used for staging liver fibrosis. Death certificates were obtained.10 copies/mL at baseline. During the follow-up, 27 patients died, with a mortality rate of 28.2/1000 person-year. Cocaine was used by 48% of those who died . Those who died were more likely to use cocaine  and have more advanced liver fibrosis , adjusting for age, gender, CD4 cell count and HIV-viral load at baseline and over time. Among the HIV mono-infected participants, cocaine users were 5 times more likely to die  than participants who did not use cocaine.Participants were 65% men, 69% Black and 81% were on ART at recruitment. Cocaine was used by 32% of participants and 29% were HIV/HCV co-infected. Mean age was 46.9 ± 7.7 years, mean CD4 cell count was 501.9 ± 346.7 cells/μL and mean viral load was 2.75 ± 1.3 logCocaine use and liver fibrosis are strong and independent predictors of mortality in HIV infected and HIV/HCV co-infected adults. Effective interventions to reduce cocaine use among people living with HIV (PHLW) are needed. As antiretroviral therapy (ART) increases the lifespan of people living with HIV (PLWH), the risk for liver cirrhosis and liver carcinoma increases . Liver dDespite promising new interventions to address HCV co-infection , cocaineLiver injury, regardless of the underlying etiology, is associated with hepatic fibrosis, a wound healing process characterized by excessive deposition of the extracellular matrix and scar formation , leadingin vitro studies  /[PLT (102 (U/L)] . Liver f2 (U/L)] . At a cu2 (U/L)] .Drug, alcohol and tobacco use questionnaires were administered by trained and certified interviewers. The drug use questionnaires were validated against blood metabolites and urine screening  and descUrine was collected under observation at each study visit. The American Biomedica Rapid Drugs kits were used to confirm self-report for cannabinoids, cocaine, methadone, methamphetamines and opiates.Death certificates were obtained from the State of Florida Department of Vital Statistics through a research agreement to verify time and cause of death and to determine whether the death was HIV-related.To characterize the population, we used descriptive statistics that were expressed as mean ± standard deviation (SD) or as percent of population. Significance was defined as P<0.05. The Cox proportional hazard model was employed to derive adjusted hazard ratios after testing for non-violation of the proportionality assumption in each case. We confirmed this by plotting the log-negative-log of the Kaplan-Meier estimates of the survival function versus the log of time using ‘R’ statistical software, version 3.1.1. The resulting curves were parallel . The comh (t)=h0 (t) × exp {b1 × 1+b2 × 2+...+bpxp} where the hazard function is represented by h (t) and is determined by a group of covariates  whose effect is measured by the size of the individual coefficients  and t is the survival time. All statistical analysis were performed using SAS statistical software, version 9.4. Mixed models were used to assess the effect of cocaine on liver fibrosis over two years. Participants in both groups were in a waiting list for ART at baseline, depending on when they were diagnosed, but were referred for treatment during participation. The great majority received treatment within one year of starting in the study, as ART became more accessible in Miami-Dade. The analyses were adjusted for viral load at baseline and over time, because viral load control was a stronger predictor of disease progression and mortality than just receiving ART. All the analyses were also adjusted for age, gender, CD4 cell count and HIV-viral load at baseline and over time.10 copies/mL at baseline . Fewer cocaine users received ART at baseline  and they had higher HIV viral load than the participants who did not use cocaine . Significantly fewer Hispanics but more African Americans used cocaine , African Americans (68%) and receiving ART (81%) at baseline. Thirty-two percent used cocaine and 29% were HIV/HCV co-infected. Mean age was 46.9 ± 7.7 years, mean CD4 cell count was 501.9 ± 346.7 cells/μL and mean viral load was 2.75 ± 1.3 logbaseline . During  cocaine .HIV/HCV co-infected and HIV mono-infected participants who used cocaine daily or more than daily, had higher FIB-4 compared to moderate cocaine users and non-users over time . In addition, daily or more than daily use of cocaine was a significant predictor of FIB-4 >1.45 in the range of moderate or more severe fibrosis over time  among HIV mono-infected participants.During the 6-year follow-up, 27 patients died, 13 of whom (48%) reported cocaine use during the study. The mortality was higher among cocaine users  compared to non-users . Twelve participants died among those who were HIV/HCV co-infected  and 15 participants among those who were HIV mono-infected . The specific rate of mortality for participants who were HIV/HCV co-infected was 12.32/1000 patient-year. P=0.003, (Liver disease is a predominant cause of morbidity and mortality in HIV infection –4. This P=0.003, , which iIn previous reports , 31, we The faster liver disease progression and elevated mortality observed among cocaine users compared with non-users in this study may be due to cocaine users having higher viral load and lower use of ART, but also to direct damage to the liver by cocaine, which may range from just elevated hepatic enzymes to hepatic necrosis precipitated by high cocaine doses . In animAnother unique finding was that faster liver disease progression and higher rate of cocaine-related mortality occurred in both HIV/HCV co-infected and HIV mono-infected participants. While the effect of high frequency of cocaine use was significantly associated with the progression of liver disease and mortality in HIV mono-infected participants, HCV co-infection was most likely a stronger driver of liver fibrosis among the HIV/HCV co-infected participants .In this study we used a non-invasive measure of liver fibrosis, the FIB-4 index, which has been suggested as an alternative to liver biopsy in PLWH, due to significant limitations such as sampling errors, observer variation and the risk for severe complications such as severe bleeding , 63. TheThis study determined the associations between cocaine use, the progression of liver disease and fibrosis, and mortality among HIV/HCV co-infected and HIV mono-infected PLWH. While the participants had a long-term follow-up, the study was observational and therefore it is not possible to draw causative inferences. Our findings are also limited by the specific characteristics of the population: people living with HIV and substance abuse in Miami, which may be very different than other populations of PLWH nationally and internationally.Cocaine use increased the likelihood of progression of liver fibrosis and death in HIV mono-infected and HIV/HCV co-infected participants. Findings from this study suggest that cocaine use in PLWH has deleterious effects on viral control, progression of liver disease and survival and that the detrimental impact of cocaine use remains even when co-infection with HCV is not present. These results suggest the need to investigate potential mechanisms of action of HIV and cocaine on liver fibrosis and survival in human studies and for developing effective intervention programs in people living with HIV to increase access and adherence to treatment, reduce progression of liver fibrosis and prevent early death.


Omphisa fuscidentalis. In both in vivo and in vitro experiments, we here measured the expression levels of genes which were affected by juvenile hormone analogue (JHA: S-methoprene) and 20-hydroxyecdysone (20E) in diapausing O. fuscidentalis larvae. Corresponding mRNA expression changes in the subesophageal ganglion (SG) and prothoracic gland (PG) were evaluated using qRT-PCR. The data showed similar response patterns of JH receptor gene (OfMet), diapause hormone gene (OfDH-PBAN), ecdysone receptor genes (OfEcR-A and OfEcR-B1) and ecdysone inducible genes . JHA induced the expressions of OfMet and OfDH-PBAN in both SG and PG, whereas ecdysone receptor genes and ecdysone inducible genes were induced by JHA only in PG. For 20E treatment group, expressions of ecdysone receptor genes and ecdysone inducible genes in both SG and PG were increased by 20E injection. In addition, the in vitro experiments showed that OfMet and OfDH-PBAN were up-regulated by JHA alone, but ecdysone receptor genes and ecdysone inducible genes were up-regulated by JHA and 20E. However, OfMet and OfDH-PBAN in the SG was expressed faster than OfMet and OfDH-PBAN in the PG and the expression of ecdysone receptor genes and ecdysone inducible genes induced by JHA was much later than observed for 20E. These results indicate that JHA might stimulate the PG indirectly via factors (OfMet and OfDH-PBAN) in the SG, which might be a regulatory mechanism for larval diapause termination in O. fuscidentalis.In insects, juvenile hormone (JH) and 20-hydroxyecdysone (20E) regulate larval growth and molting. However, little is known about how this cooperative control is terminating larval diapause especially in the bamboo borer,  The 20E is transformed from ecdysone, produced in the prothoracic glands (PG) and triggers larval molting and metamorphosis with the larval-pupal transition [Helicoverpa spp and regulates female reproductive maturation in adult insects [Drosophila melanogaster) and later confirmed in other insects [Insect molting and metamorphosis are regulated by the interplay of two hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) . In inseansition . PTTH stansition . The priansition . In immaansition , 6. The ansition . This reansition . Moreove insects –12. The  insects . Circula insects . The lig insects . The pro insects .in vivo or in vitro [Tribolium castaneum, Met plays a similar key role in JH action during the larval-pupal metamorphosis and precocious development of adult structures, indicating that Met is involved in antimetamorphic JH signaling [The mechanism mediating the JH response is a fascinating question. Recently, some studies have reported that JH could induce the transcription of a large number of genes in vitro –19, and in vitro . Howeverin vitro . JH bindin vitro , 22. Theignaling , 24.D. melanogaster [B. mori [T. castaneum [In holometabolous insects, Br-C are key players in the JH regulation of metamorphosis and is a transcription factor that directs pupal development . In the nogaster , B. mori[B. mori , 31, andastaneum , 32.Diatraea grandiosella, the JH titer remains elevated throughout diapause and the diapause can be terminated only when the JH titer drops [Ostrinia nubilalis, the JH titer is high in early diapause but then declines and remains low throughout the remainder of diapause [Larval diapause is generally known as an arrested period of post-embryonic development characterized by a major shutdown in metabolic activities and is under the control of hormonal interactions . During er drops . In somediapause .Omphisa fuscidentalis), a univoltine lepidopteran insect, is found in Northern Thailand, Laos and Myanmar [O. fuscidentalis, the ecdysteroid titer in the hemolymph was found to be very low [O. fuscidentalis larvae induced pupation by increasing the ecdysteroid titer in hemolymph [O. fuscidentalis larvae, indicating that the brain is not the primary target of the JHA [O. fuscidentalis were treated with JHA, the secretory activity of PG was increased at 10 days after JHA treatment [O. fuscidentalis by increasing levels of ecdysteroid in the hemolymph , correlated with diapause hormone gene (OfDH-PBAN) mRNA level during the development of O. fuscidentalis [OfDH-PBAN transcript may be involved in regulating larval diapause termination. Although the effect of JHA on the expression of DH-PBAN genes has not been demonstrated in other insect’s tissues, our preliminary experiment showed that the expression of OfDH-PBAN mRNA in SG was much higher than PG (data not shown). This result indicated that DH-PBAN gene was synthesized primarily in SG [OfMet, OfDH-PBAN, ecdysone receptor genes (OfEcR-A and OfEcR-B1) and ecdysone inducible genes  in SG and PG both in vivo and in vitro. The knowledge of expression pattern of OfMet, OfDH-PBAN, ecdysone receptor genes and ecdysone inducible genes may help us to better understand the mechanisms of larval diapause termination in O. fuscidentalis.The bamboo borer . In addition, the field studies in this research did not involve endangered or protected species. No specific permissions were required for the bamboo forest where the bamboo borers were collected. Diapause in this O. fuscidentalis population usually lasts from September to June [et al. (1999).From November to December 2014, larvae of  to June . Therefoet al. . The selected dose was used as a critical dose in all experiments. The JHA  was dissolved in acetone  and stored at -35°C as a stock solution. The stock solution was diluted to 0.5 μg per 5 μL with acetone, and a 5 μL aliquot was applied to the dorsal abdomen of individual diapausing larvae using a 50 μL microsyringe (N = 700) [The doses of JHA and 20E (0.5 μg/larva) were able to induce pupation according to the established works by Singtripop N = 700) . IndividN = 700) .-1 and stored at -35°C until use. The stock solution was diluted to 0.5 μg per 5 μL in DW, and diapausing larvae were injected with a 5 μL aliquot through the second proleg (N = 700) [The 20-hydroxyecdysone , was dissolved in distilled water (DW) at a concentration of 1 mg mLN = 700) . ControlTotal RNA was isolated from SG and PG using RiboZol RNA Extraction reagent  according to the manufacturer’s instructions. Before cDNA synthesis, the RNA was treated with RNase-free DNaseI  to eliminate contaminating DNA. A quantity of 1 μg of total RNA was used to generate first-strand cDNA with an oligo-dT primer and M-MuLV-Reverse Transcriptase (Fermentas).-1) was prepared in EtOH. The solution was evaporated in air and resuspended in Grace’s insect cell culture medium . All equipment used in the JHA experiments was coated with 1% solution of polyethylene glycol. For the 20E experiments, the stock solution (0.02 μg μL-1) was prepared in DW. Both JHA and 20E stock solutions were serially diluted in culture medium to the appropriate working concentrations. In brief, the diapausing larvae were anesthetized on ice for 30 min. The SG and PG were dissected and rinsed 5 times in ice-cold Ringer’s solution . The tissue was rinsed again in culture medium five times. The hormones were then added to 50 μL medium containing 1 lobe of SG and PG in 96-well culture plates  and were incubated at 25°C with gentle shaking. For the controls, equal volumes of the medium without JHA or 20E were added. Total RNAs from SG and PG in both hormone treatments and control experiments were extracted at the same time points after incubation.A diluted stock solution of JHA (0.02 μg μLOfRpL3), is the most stable and constant in B. mori nervous system [OfRpL3 expression, and found that RpL3 showed the constant expressions in PG and SG of O. fuscidentalis [OfRpL3 mRNA was used as an endogenous control. Quantitative real-time PCR was conducted using the SensiFASTTM SYBR® No-Rox Kit  and an iCycler iQ5™ Real-Time PCR Detection System . One reaction contained 1 μL of template cDNA sample and 0.2 μM primers in a final reaction volume of 20 μL. The thermal cycling parameters were 95°C for 2 min, followed by 40 cycles at 95°C for 5 sec, 60°C for 10 sec, and 72°C for 20 sec. After qRT-PCR, the absence of undesired by-products was confirmed by an automated melting curve analysis and agarose gel electrophoresis (1.5% w/v) of the PCR product. Also, amplified PCR products were purified by using a GeneJET Miniperp Kit (Fermentas) and DNA sequencing was performed by an ABI PRISM Bigdye Terminator (version 3.1) cycle Sequencing Kit  and automated DNA sequencer . The amplification efficiency for both the reference and target genes was analyzed. The relative expression levels of OfMet, OfDH-PBAN, ecdysone receptor genes (OfEcR-A and OfEcR-B1) and ecdysone inducible genes  from samples with different treatments were estimated by a comparative CT method (ΔΔCT) for relative quantitation of gene expression. The CT (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the background level. The dynamic range of the target genes and normalizer (OfRpL3) were determined. After normalization with OfRpL3 [i.e., ΔCT = COfMet, OfDH-PBAN, ecdysone receptor genes or ecdysone inducible genes)T (—COfRpL3)T . All results of qRT-PCR were analyzed using Pfaffl’s mathematical model [The primers used for amplifying cDNA fragment are listed in s system . For nordentalis . Therefolibrator  . For the in vivo datasets, the effects of JHA and 20E on OfMet, OfDH-PBAN, ecdysone receptor genes and ecdysone inducible genes expression were examined separately for SG and PG. To determine whether the relative expressions differ among larval and pupal development stages, we used the one-way analysis of variance (ANOVA) to analyze each expression. The means of relative expression levels from three replicates was used as a dependent variable and the stages of larval and pupal development was used as an independent variable. For the JHA dataset, the stages of larval development were categorically independent variable with 11 levels—day 0, 2, 4, 6, 8, 10, 12 after the treatment and four pupal stages (G0-G3). For the 20E dataset, the larvae went into pupal stages sooner than those in the JHA treatment. Therefore, the stages of larval development in the 20E treatment contained 10 levels—day 0, 2, 4, 6, 8, 10 after the treatment and four pupal stages (G0-G3). The normality of dependent variable residuals and the homogeneity of variances of the data were checked to ensure that the assumptions of ANOVA were met. When the results of ANOVA showed that there were differences in the relative expression levels among the stages of larval and pupal development, a Tukey’s HSD test was performed to obtain the information on which means are significantly different from each other at a significance level of 0.01. Data are presented as the mean ± SE.In this study, there were two main parts of the experiments, including in vitro dataset, in addition to JHA and 20E treatment, the data on combined effects of JHA+20E were also analyzed. The means of relative expression levels from three replicates was used as a dependent variable and the time after the treatment application was used as a categorically independent variable with 9 levels—0, 0.5, 1, 4, 8, 12, 16, 20 and 24 hours after the treatment application. The analyses were performed in the same fashion as the in vivo datasets.For the OfMet, OfDH-PBAN, ecdysone receptor genes and ecdysone inducible genes in both SG and PG , however it later decreased on day 10 and abruptly dropped to a minimum level until G3 stage . Expression of OfDH-PBAN in both SG and PG then abruptly dropped to a low level at G0-G3 stage (P < 0.01) (P < 0.01) (We induced pupation by treating diapausing larvae with JHA (the treated diapausing larvae entered pupation stage at Day 12) and examined the effect of JHA on expression levels of G and PG . After tG3 stage . In addiG3 stage . In the G3 stage . By cont < 0.01) . In addi < 0.01) .OfMet and OfDH-PBAN in SG and PG. JHA also induced the expression levels of ecdysone receptor genes and ecdysone inducible genes in PG during pupal stage .These results indicated that JHA significantly increased expression levels of OfMet, OfDH-PBAN, ecdysone receptor genes and ecdysone inducible genes in both SG and PG (OfMet and OfDH-PBAN in both SG and PG were low from day 0 to G3 stage and they were not significantly different when compared with controls (P < 0.01) (P < 0.01)  . Further < 0.01) .OfMet, OfDH-PBAN, ecdysone receptor genes and ecdysone inducible genes were examined under these experimental conditions.To understand how hormonal mechanism on termination of larval diapause is regulated by JHA, the SG was cultured in Grace’s medium containing both JHA at 0.1 μg/50 μl and 20E at 0.1 μg/50 μl for different continuous time exposures. The expression patterns of OfMet mRNA levels were low at 0 h, and then rapidly increased to the maximal value at 0.5 h , followed by a decline from 1 to 24 h   (P < 0.01), followed by a large decrease at 4 h and remained steady at a low level until 24 h (P < 0.01) and decreased at 8–24 h after 20E application  . By cont < 0.01) . In the  > 0.01)  whereas til 24 h . The rellication . These rOfMet and OfDH-PBAN transcripts showed temporal patterns similar to those obtained in response to JHA  (P < 0.01)  together affected gene expressions as a function of time. When SG were co-cultured with 0.1 μg/50 μl of JHA and 20E,  < 0.01) , while e < 0.01) .OfMet, OfDH-PBAN, ecdysone receptor gene and ecdysone inducible gene expressions in PG, the PG was incubated in Grace’s medium containing both 0.1 μg/50 μl of JHA and 0.1 μg/50 μl of 20E as a function of time. In the presence of JHA alone, the results showed that the relative expression of OfMet was low at 0 h, rapidly increased at 0.5 h, peaked at 1 h  and then decreased at 4–24 h, while OfDH-PBAN was low between 0 and 1 h, then increased suddenly to a maximum level at 4 h , followed by a decrease at 8 h and remained at low levels from 12–24 h (P < 0.01), followed by a decline at 12–24 h (P < 0.01), then declined and remained steady at a low level until 24 h after JHA addition (OfMet and OfDH-PBAN mRNA levels remained at a low level at 0–24 h and were not significant when compared with the controls (P < 0.01) and decreased at 1 h. Expression of ecdysone receptor genes then declined abruptly to low levels until 24 h . Thereafter, it decreased at 4 h and then remained at low levels from 8–24 h , followed by a decline at 8–24 h after the application of 20E  (P < 0.01)  , while t < 0.01) .in vivo and in vitro experiments results displayed similar patterns of expressions of OfMet, OfDH-PBAN, ecdysone receptor genes and ecdysone inducible genes in SG and PG. Interestingly, JHA induced the expression of OfMet and OfDH-PBAN in SG and PG whereas 20E had no significant effect on expression of OfMet and OfDH-PBAN in both tissues, indicating that 20E might not be involved in the up-regulation of OfMet and OfDH-PBAN genes expression in SG and PG but both genes were directly up-regulated by JHA only. Our results of OfMet expression patterns in bamboo borer were similar to that of BgMet in whole body of Blattella germanica when treated with juvenile hormone [Met was shown as a rapid response to JHA, indicating that JH itself may plays a crucial role in regulating the expression of its receptor [T. castaneum, D. melanogaster and B. mori) maintain continuous expression of Krüppel homolog 1 (Kr-h1), which is an early JH-response gene via Met, and Kr-h1 inhibits metamorphic changes [Kr-h1 expression via Met in the pupal stage of D. melanogaster [T. castaneum [Br-C and formation of second pupae. Moreover, JH induced the expression of ecdysone inducible genes such as Br-C [E75 [HR3 [Met and Kr-h1 could be involved in mediating JH signals. Therefore, it is possible that JH affects the expression of 20E-related genes via Met and Kr-h1 and Met plays a role in mediating termination process of larval diapause in O. fuscidentalis.In our study, both  hormone . Recentl hormone , 48. Thireceptor . In natu changes , 50, 51.astaneum , which i as Br-C , 28, E75r-C [E75 , 53 and E75 [HR3 . This coet al. (2015), the expression of DH-PBAN was generally observed in neural tissues including the brain, SG, the thoracic ganglion and the abdominal ganglion. The highest expression was shown in SG. To study the signaling pathway of JHA on diapause termination in the bamboo borer, and involvement of PG as a source for the elevation of ecdysteroid, we focused on DH-PBAN expression in PG. Without stimulating PG with JHA, we could measure the expression levels of DH-PBAN in PG of natural larvae and pupae DH-PBAN mRNA may originate from terminals of the DH-PBAN neurons with somata in the SG. In addition, the DH-PBAN neurons may even innervate the PG in the bamboo borer. Thus, we would like to propose an alternative hypothesis that the DH-PBAN mRNA, originally expressed from the terminals of DH-PBAN SG neurons, might be present in the isolated PG samples.According to Suang OfDH-PBAN, the specific primers for OfDH-PBAN were designed  and used to amplify the gene in PG. A single band was clearly evident in the gel after electrophoresis OfDH-PBAN nucleotide sequence OfDH-PBAN was expressed in PG.To clarify the specificity of OfMet, but OfDH-PBAN was also upregulated by JHA. It was clear that OfMet and OfDH-PBAN in the SG were expressed much faster than OfMet and OfDH-PBAN in PG, suggesting that one of the functions of JH in the termination of larval diapause could be to directly stimulate OfMet and OfDH-PBAN expressions in the SG. Nevertheless, the mechanism of DH to activate PG in releasing ecdysone, and then stimulate larvae to break the diapause is still unknown. Our preliminary experiment showed that exogenous DH could also break larval diapause of O. fuscidentalis by increasing levels of ecdysteroid in the hemolymph . This result was also in agreement with a previous study obtained from Helicoverpa spp. [et al. (2007) found that the Bombyx diapause hormone receptor (BmDHR) was expressed in the PG, and the FXPRL-amide peptides were able to induce ecdysteroidogenesis in the PG through the activation of BmDHR [OfDH-PBAN genes might be one of the hemolymph factors to terminate larval diapause, after being synthesized from the SG and released into the hemolymph in O. fuscidentalis. According to our results, OfDH-PBAN mRNA was also expressed in PG. In this study, we focused on mRNA expression to display the correlation at transcription level between JHA, DH-PBAN and diapause termination in the bamboo borer. The effects of JHA on DH-PBAN at a protein expression level will need to be investigated to reaffirm the action of JHA and DH-PBAN.Not only rpa spp. . Moreoveof BmDHR . TherefoO. fuscidentalis, 20E is tightly involved in the termination of the larval diapause by increasing the ecdysteroid titer in the PG directly [in vitro that the EcR genes were directly stimulated by 20E in the larvae of Lepidopterans such as M. sexta [P. interpunctella [B. mori [EcR genes [Br-C [E75 [HR3 [EcR homologous gene in the M. sexta epidermis which JH prevented the 20E-induced metamorphic switching by regulating the induction of EcR by 20E [OfE75 expression was similar to the previous reports for D. melanogaster [M. sexta [O. fuscidentalis larvae were treated with 1 μg JHA, the ecdysteroid titer increased in the hemolymph. This indicated that the brain was not the target tissue for JHA [OfMet and OfDH-PBAN in SG and then acting on PG to release ecdysone. Thus, the ecdysteroids produced by the PG might have up-regulated the OfEcR genes in prothoracic gland cells, which in turn causes induction of ecdysone inducible genes and termination of larval diapause in O. fuscidentalis.In directly . This coM. sexta , P. inteunctella , and B. [B. mori . FurthercR genes , 62, 63 es [Br-C , 64, 65,r-C [E75 , 66–69 aE75 [HR3 , 71. MeaR by 20E . The effnogaster  and M. sM. sexta , 74. Aft for JHA . In addi for JHA . Our resin vitro. These results indicate that JHA did not directly up-regulate the expressions of ecdysone receptor genes and ecdysone inducible genes in PG, but JHA could stimulate ecdysone synthesis in PG via OfMet and OfDH-PBAN in SG, which then could feed back on the glands to induce the synthesis of these genes within the gland.In addition, the simultaneous treatment with JHA and 20E displayed combined effects of JHA and 20E. The increase in the expressions of ecdysone receptor genes and ecdysone inducible genes in PG incubated with both JHA and 20E was higher than PG incubated in medium containing either JHA or 20E. This observation suggested that the presence of both hormones increased expression levels of these genes in PG. However, the expression levels of ecdysone receptor genes and ecdysone inducible genes were induced by JHA much later than observed for 20E in vivo experiments, the expression levels of OfMet and OfDH-PBAN before entering the pupal stage and the expression levels of ecdysone receptor genes and ecdysone inducible genes in the pupal stage may be associated with an increment of JH and 20E titer in the hemolymph. In O. fuscidentalis, the hemolymph ecdysteroid concentration is low during larval diapause and increases prior to the pupal stage, thereby presumably stimulating pupal metamorphosis [O. fuscidentalis has not been determined. A previous study has shown that the expression level of juvenile hormone binding protein (JHBP) encoding gene in the fat body was high in the diapause period and low from the late-diapause until pupation [In addition, for orphosis . Howeverpupation . Change O. fuscidentalis has the longest larval diapause period to adapt themselves to the natural habitats [O. fuscidentalis while a high level of juvenile hormone maintains larval stage in other lepidopteran insects. The finding in this study may help us to understand the relationship between JH and 20E on termination of larval diapause in O. fuscidentalis, which JH might play a crucial role in regulating ecdysteroid signaling pathway in this mechanism. Further studies to analyze expression level of the enzymes, which are related with ecdysteroid production are needed to elucidate the hormonal regulation of PG activity and of the enzymes which are related with ecdysteroid production. of the enzymes which are related with ecdysteroid production. of the enzymes which are related with ecdysteroid production. the unique regulatory mechanism of O. fuscidentalis larval diapause.Amongst insects, habitats . JuvenilS1 FigOfRpL3). Expression is relative to the gene expression in diapausing larvae from October . Each value is the mean ± SEM of three independent experiments. Means with different letters indicate a significantly difference The results are expressed as the relative expression after normalization against endogenous ribosomal protein mRNA ((PDF)Click here for additional data file.S2 FigOfDH-PBAN mRNA by using primers OfDH-SPF and OfDH-SPR. M, marker for DNA molecular weight. (B) Nucleotide sequence and the deduced amino acid sequence of the PCR products.(A) PCR product of (PDF)Click here for additional data file.


Disrupted-In-Schizophrenia-1 (DISC1), a well-accepted genetic risk factor for mental illness, display abnormal behaviours in response to stress, but the mechanisms through which DISC1 affects stress responses remain poorly understood. Using two lines of zebrafish homozygous mutant for disc1, we investigated behaviour and functioning of the hypothalamic-pituitary-interrenal (HPI) axis, the fish equivalent of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we show that the role of DISC1 in stress responses is evolutionarily conserved and that DISC1 is essential for normal functioning of the HPI axis. Adult zebrafish homozygous mutant for disc1 show aberrant behavioural responses to stress. Our studies reveal that in the embryo, disc1 is expressed in neural progenitor cells of the hypothalamus, a conserved region of the vertebrate brain that centrally controls responses to environmental stressors. In disc1 mutant embryos, proliferating rx3+ hypothalamic progenitors are not maintained normally and neuronal differentiation is compromised: rx3-derived ff1b+ neurons, implicated in anxiety-related behaviours, and corticotrophin releasing hormone (crh) neurons, key regulators of the stress axis, develop abnormally, and rx3-derived pomc+ neurons are disorganised. Abnormal hypothalamic development is associated with dysfunctional behavioural and neuroendocrine stress responses. In contrast to wild type siblings, disc1 mutant larvae show altered crh levels, fail to upregulate cortisol levels when under stress and do not modulate shoal cohesion, indicative of abnormal social behaviour. These data indicate that disc1 is essential for normal development of the hypothalamus and for the correct functioning of the HPA/HPI axis.Psychiatric disorders arise due to an interplay of genetic and environmental factors, including stress. Studies in rodents have shown that mutants for  Phenotypes are shaped throughout the life-course by a complex interplay between genes and the environment. When homeostasis is threatened by environmental stress, animals respond adaptively by altering their metabolism, physiology and behaviour. These adaptive responses are co-ordinated by the hypothalamic-pituitary-adrenal (HPA) axis . ActivatDisrupted-In-Schizophrenia-1 (DISC1), was originally identified at a chromosomal translocation breakpoint in a single Scottish family, in which a high proportion of family members suffered from mental illness  axis in fish.Expression studies in primates and mice have shown that thalamus , a smallervation . We prev embryos . We therdisc1 (L115X and Y472X) and analysed baseline and stress-responsive behaviours in the adult. We investigated the developmental origin of mutant behavioural abnormalities, and show that disc1 is essential for normal development of the early hypothalamus and HPI axis function.To address this hypothesis, we utilised two lines of zebrafish harbouring nonsense mutations in N-terminal head domain of DISC1 /2–3 days post-fertilisation (dpf)) revealed that expression is most prominent in the basal part of the brain, in particular the hypothalamus  a conserved paired-like homeodomain transcription factor  larvae. In both lines, rx3 is altered in disc1 mutants in comparison to wild type siblings  , an M-phase marker whose expression correlates with proliferating progenitors in the embryonic zebrafish hypothalamus  . In L115siblings , but frosiblings . Transveype fish . By contentricle  legend. thalamus . At 24 hsiblings . At 3 dpsiblings . Togetherx3+ progenitors in the tuberal hypothalamus give rise to VMN neurons that express the nuclear receptor, ff1b   and to n (pomc) ,54. We tnal cues .ff1b is first detected in the developing hypothalamus at 24 hpf  defines Arc-like neurons in the hypothalamus. In wild type zebrafish, hypothalamic b+ cells . Analysi mutants . In both animals .disc1 affects corticotropin releasing hormone (crhb)-expressing neurons, a population that is specified in an rx3-independent manner  and pomc-positive neurons  are preferentially increased in number in disc1 mutant fish. In summary, our studies suggest that in zebrafish, disc1 is required for proliferation of rx3-positive progenitors, with loss of disc1 function leading to premature differentiation and early excessive production of ff1b-positive neurons.The observed anxiety-like behaviours and failure to activate appropriate stress responses in the adult recesses ,51. Studmaturely . Our stuve cells ,51, and rogramme . This fuSF1/NR5A1 to anxiety. Central nervous system-specific knockout of Sf1 in mice leads to increased anxiety-like behaviours  or a control solution (water) were pipetted into the centre of the fish container and movements were tracked for 10 min. Alarm substance was extracted according to the method of Schirmer and colleagues  and usedThe open field and dark-light test tank was 25 x 15 x 15 cm and filled to 4.1 L, whilst the trapezoid tank diving test tank was 23.5 x 6.2 x 13.5 cm and filled maximally. The open field and tank diving tanks were bare, whilst the light-dark tank was half covered in a black opaque material on all sides. Fish were acclimated to the open field tank for 1 h prior to filming, whilst filming in the light-dark and tank diving tests began immediately after the fish was transferred to the tank. Light time is the percentage of time each fish spends in the light compartment, whilst bottom dwell is the percentage of time a fish spends in the lower half of the tank diving test tank.Nearest-neighbour distance (NND) was quantified based on the formula provided by Miller & Gerlai . Brieflyet al. , pomc . For analysis of disc1, eyes were removed after fixation. After staining, larvae were re-fixed then transferred to 30% sucrose for cryosectioning. Specimens were mounted in OCT and 12 μm thick transverse sections through the entire forebrain, were serially collected. Sense probes were routinely used as controls.Whole-mount rotocols . The folols , pomc , crhb . This enabled accurate matching of sections from wild type and mutant siblings. Note that in some cases, optic vesicles were displaced upon cryosectioning. Width across the lateral recess, in which et al.  at 1:1000, as described by Muthu et al. , and mouImages of whole-mount and sectioned zebrafish were acquired using an Olympus BX60 microscope using Q Capture Pro 7.0 (QImaging). Images were processed using Adobe Photoshop CC 2014.t tests, for comparisons between two samples, whilst samples classified by two or more different types of treatment were analysed by Analysis of Variance (ANOVA) and post hoc analysis via Tukey’s test. For quantitative RT-PCR, ΔCT values for disc1 were calculated relative to ef1a for each sample. Fold change was calculated for each sample relative to the mean of the control group (wild type). Absorbance readings of cortisol standards were used to create a standard curve. Cortisol concentrations of experimental samples were determined by interpolation using a 4-parameter non-linear regression curve fit. In all cases, standard error of the mean is reported and *P < 0.05, **P < 0.01, ***P < 0.001.Statistical analysis and graphics were created in ‘R’ Version 3.3.0 . Data weHMG online.Supplementary DataClick here for additional data file.


The intracerebral hemorrhage (ICH) score is a commonly used prognostic model for 30-day mortality in ICH, based on five independent predictors . Use of oral anticoagulants (OAC) is also associated with mortality but was not considered in the ICH score. We investigated (a) whether the predictive performance of ICH score is similar in OAC-ICH and non-OAC-ICH and (b) whether addition of OAC use to the ICH score increases the prognostic performance of the score.We retrospectively selected all consecutive adult non-traumatic ICH cases . Mortality at 30 days was recorded. Using univariable and multivariable logistic regression, association between OAC use and 30-day mortality was tested. Then (a) we computed receiver operating characteristic (ROC) curves for ICH score and determined the area under the curve (AUC) in OAC-ICH and non-OAC-ICH. Then (b) we created a New ICH score by adding OAC use to the ICH score. We calculated correlation between 30-day mortality and ICH score, respectively, New ICH score using Spearman correlation test. We computed ROC curves and calculated the AUC.p < 0.001), corrected for the five predictors of the ICH score. The ICH score performed slightly better in non-OAC-ICH (AUC 0.840) than in OAC-ICH (AUC 0.816), but this difference was not significant (p = 0.39). The ICH score and the New ICH score were both significantly correlated with 30-day mortality . The AUC for the ICH score was 0.837, for New ICH score 0.840. This difference was not significant.We analyzed 1,232 cases, 282 (22.9%) were OAC related ICH. Overall, 30-day mortality was 39.3%. OAC use was independently associated with 30-day mortality (OR 2.09, 95% CI, 1.48–2.95; The ICH score is a useful tool for predicting 30-day mortality both in patient who use and patients who do not use OAC. Although OAC use is an independent predictor of 30-day mortality, addition of OAC use to the existing ICH score does not increase the prognostic performance of this score. Spontaneous non-traumatic intracerebral hemorrhage (ICH) constitutes about 10–15% of all strokes and has a high mortality of approximately 40% at 1 month –8; the mUp to 25% of all ICH is associated with the use of oral anticoagulants (OAC), and this percentage increases with age . OAC useTherefore, we aim to determine the prognostic performance of ICH score for 30-day mortality in OAC-ICH and non-OAC-ICH in a large retrospectively collected multi-center cohort of ICH patients. We also aim to test whether OAC use is an independent predictor of outcome in ICH patients and if addition of OAC use to the ICH score increases the prognostic performance of the score.We retrospectively collected all consecutive adult (≥18 years) patients with an imaging-confirmed (CT- or MRI-scan) non-traumatic ICH, seen in the emergency department, inpatient or outpatient clinic, in three hospitals of South-Limburg, the Netherlands, from January 2004 to December 2009. Patients were selected using diagnosis-treatment codes retrieved from hospital Medical Registration Archives complemented with hospital stroke registries. Recurrent ICH cases were included.Exclusion criteria were traumatic ICH or non-parenchymal hemorrhage , hemorrhagic transformation of ischemic stroke, brain tumor associated hemorrhage, or hemorrhage with a known vascular malformation. Patients with non-accessible charts and/or scans were also excluded.The medical ethical committee of Maastricht University Medical Center approved the study.We recorded age, sex, GCS at admission, current OAC use, and first INR at admission. OAC-ICH was defined as the occurrence of an ICH while on treatment with oral vitamin K antagonists at admission. Hemorrhage properties were measured on first CT- or MRI-scan: supra- or infratentorial, intraventricular extension, and ICH volume. ICH volume was calculated using the ABC/2 method , 17. SurThe ICH score (0–6) was calculated as described by Hemphill et al. . One poiWe then created a New ICH score by adding an additional point for the use of OAC. The maximum New ICH score was 7.Categorical variables are presented as frequencies with percentage, continuous and ordinal variables as mean with SD or median with quartiles, depending on the distribution of the data.First, we investigated whether OAC use is a predictor of 30-day mortality. We performed univariable logistic regression analysis with 30-day mortality as outcome variable and the variables in the ICH score  and OAC use as predictors. We then performed a multivariable logistic regression using the same variables to see whether OAC use is an independent predictor. There were no problems with multicollinearity.Second, we investigated ICH score in both OAC-ICH and non-OAC-ICH. Correlation between ICH score and 30-day mortality was determined in patients with OAC-ICH and in patients with non-OAC-ICH, using Spearman correlation test. We computed receiver operating characteristic (ROC) curves and determined the area under the curve (AUC) in OAC-ICH and non-OAC-ICH.Finally, we tested the New ICH score: Spearman correlation was determined between ICH score and mortality, and between New ICH score and mortality. We computed ROC curves and determined and compared the AUC for both ICH score and New ICH score. We also computed the difference in net benefit between ICH score and New ICH score at a threshold of 90% for predicted probability of mortality, which indicates how many more true positive mortality classifications can be made with the same number of false positive classifications.p value of <0.05 was considered significant.Statistical analysis was conducted using IBM SPSSS statistics 22.0. Analysis of net benefit was performed in R version 3.1.1. A n = 484). There were 282 (22.9%) OAC-ICH. Median INR in these patients was 3.5 (IQR 2.7–4.4).We recorded 1,276 consecutive patients with a spontaneous non-traumatic ICH. Of those, 44 (3%) were excluded due to incomplete data on ICH score or 30-day mortality status, which left 1,232 patients in the analyses. Table p < 0.001).Table p < 0.001). 30-Day mortality in non-OAC-ICH was 329 (34.6%) and in OAC-ICH 155 .The median ICH score in non-OAC-ICH and OAC-ICH are 1 (IQR 1–3) and 2 (IQR 1–3), respectively  than in OAC-ICH , but this difference was not significant (p = 0.39).There was a positive correlation between ICH score and mortality in non-OAC-ICH, Spearman’s rho 0.57, An ICH score of 0 was present in 265 21.5%) patients, and 2 (0.2%) patients had the maximum ICH score of 6. Both patients with an ICH score of 6 used OAC and both died. Mortality in patients with different ICH scores is shown in Figure .5% patieUsing the new ICH score, 229 18.6%) patients had a score of 0, and 2 (0.2%) had the maximum score of 7. All patients with a New ICH score of 6 or 7 died. Figure 8.6% patip < 0.001 and 0.59, p < 0.001, respectively).The ICH score and the New ICH score were both significantly correlated with 30-day mortality . The New ICH score had a net benefit of 0.016 compared with the ICH score at a threshold probability of 90%  study that evaluated hospital mortality  have been developed –8 to preIn our study, we found OAC use to be a risk factor for 30-day mortality, independent of the five components that make up the ICH score . Despite this, adding OAC use to the ICH score did not improve the predictive value for mortality as shown in the ROC curves. However, comparison of ROC curves warrants some methodological reservations . It has per se  and only looked at vitamin K antagonists. The results can therefore not be generalized to patients with an ICH in the setting of DOAC use. Finally, it could be that anticoagulation level (INR) or reversal therapy is more important than OAC use per se . INR valIn conclusion, the ICH score remains a useful tool for predicting 30-day mortality in patient who use and patients who do not use OAC. The use of OAC is associated with higher 30-day mortality but adding OAC use to the existing ICH score did not improve the predictive value of this score.The medical ethics committee of Maastricht University Medical Center approved the study and waived the requirement for obtaining informed consent because it is a retrospective observational study.JS, FS, RH, and RO contributed to the conception and design of the study. FS, KB, and DC collected the data. FS and RH organized the database. JS and RH performed the statistical analysis. JS and RH wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Limited data are available regarding the impact of the potential validation of the Canadian Thoracic Society (CTS) guidelines recommendations in classifying patients with an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in simple and complex. The aim of the present study was to assess the CTS recommendations regarding risk stratification on clinical outcomes among patients hospitalized with an AECOPD.We developed a retrospective cohort study of patients admitted to one tertiary hospital with a diagnosis of AECOPD. The main clinical outcome was the percentage of treatment failure. Secondary outcomes were 30-day, 90-day, and 1-year readmission and mortality rate, length of stay in hospital, intensive care unit (ICU) admission rate, time to readmission, and time to death. Multivariate analyses were performed using 1-year mortality rate as the dependent measures.One hundred forty-three patients composed the final study population, most of them (106 [74.1%)] classified as complex acute exacerbation (C-AE) of COPD. C-AE patients had similar rate of treatment failure compared with simple acute exacerbation (S-AE) of COPD . There were no differences regarding the length of stay in hospital, ICU admission rate, and 30-day, 90-day, and 1-year readmission rate. C-AE patients had faster declined measures on time to death . In the multivariate analysis, after adjusting for comorbidity, lung function and previous treatment, C-AE patients had a significant higher mortality at one year ; p = 0.031).In hospitalized patients with an AECOPD, CTS classification, according to the presence of risk factors, was not associated with worse short-term clinical outcomes although it is related with long-term mortality.  Chronic Obstructive Pulmonary Disease (COPD) is a major increasing health problem among people . COPD isAlthough the infectious agents in COPD exacerbations can be viral or bacterial and the use of antibiotics in exacerbations still remains controversial -12, antiThis is a retrospective cohort study performed in one public hospital in Spain, the Complexo Hospitalario Universitario de A Coruña (CHUAC), a 1382-bed teaching hospital serving a population of 514,466 people. Division permission was obtained in order to collect data regarding impact of the quality improvement initiative at the institution as an exempt study.Eligible subjects were admitted to the hospital between January 1, 2009 and January 1, 2010 with a hospital discharge diagnosis of AECOPD . Subjects were included in the study if they were: (1) >40 years old, (2) former or active smokers (>10 packets per year), and (3) had a prior spirometry with forced expiratory volume at first second/forced vital capacity < 70 (FEV1/FVC < 70). Patients were excluded if they had radiological evidence of pneumonia. If a patient was admitted to the hospital more than once during the study period, only the first hospitalization was recorded and included in the analysis.Chart review data included demographics, comorbid conditions, basal treatment, clinical presentation, laboratory and microbiology data, and treatment during admission. Process measures previously reported, which were associated with higher risk for treatment failure or infection by virulent or resistant microbial pathogens, were recorded. Electronic health records were employed to set the date of readmission and death. Readmission and death were reviewed until 08/01/2013.Treatment failure was defined as a composite variable of one of these: 30-day readmission, 30-day mortality, the need for intubation, or prolonged length of stay . ProlongAccording to CTS COPD guidelines , the risThe main clinical outcome was the percentage of treatment failure. Secondary outcomes were 90-day and 1-year readmission and mortality rate, length of stay in hospital, intensive care unit (ICU) admission rate, time to readmission, and time to death.Descriptive baseline characteristics were presented as means (± standard deviation [SD]) for continuous variables or n (%) for categorical variables. A two-tailed sample t-test was used to compare the mean length of stay in hospital, time to death, and time to readmission between the study groups. Chi-square test or the Fisher exact test was employed to assess the statistical significance of differences in the rates of participants in the complex and simple acute exacerbation groups who did and did not die, were admitted to the ICU, or were readmitted or died at 30-day, 90-day, or 1-year. We performed a multivariate statistical analysis by logistic regression to assess the independent effect of CTS classification of mortality at one year, with the criteria largest p-value for entering variables (0.05) and smallest p-value for removing variables (0.1). Variables remaining in the multivariate analysis model that showed a p-value of 0.05 were considered significant. Differences among groups in time to death and time to readmission were assessed using the Kaplan-Meier curves and the log-rank test. A two-sided p-value of <0.05 was considered statistically significant in the analysis of proportions. All data were statistically analyzed with SPSS  and STATA .A total of 406 patients were screened, and among them 226 did not fulfill inclusion criteria (mostly because of the absence of previous lung function test or being nonsmokers) Figure .A total of 143 patients composed the final study population. Among them 106 patients (74.1%) had at least one risk factor for AECOPD (C-AE group) and 37 patients (25.9%) did not have any risk factor (S-AE group). The proportion of men was significant higher in C-AE group .Demographics, comorbidities, respiratory function, and basal treatment on the admission of the study population are summarized in Table Excluded by definition that S-AE patients did not have any CTS risk factors, there were no significant differences in the comorbidities between groups, except that C-AE patients had higher frequency of diabetes , coronary heart disease   and a non-significant tendency to have a higher Charlson index . As expected by definition, C-AE had worse pulmonary function with a mean FEV1% of 42.5 ± SD 15 vs. 67.2 ± SD 16.3; p > 0.001. Compared with S-AE, C-AE patients were treated before admission more frequently with tiotropium , combination of salmeterol/fluticasone , triple therapy , and theophylline .The distribution of CTS risk factors among C-AE is described in Table C-AE patients had similar rate of treatment failure compared with S-AE Table .There were no differences between groups regarding length of stay in hospital, ICU admission rate, 30-day, 90-day, and 1-year readmission rate to patients without risk factors. Compared to S-AE, C-AE patients had faster declined measures on time to death , without differences in time to first readmission .Microbiological samples were taken in a similar percentage of patients among each group. Compared with S-AE patients, more sputum cultures were positive among C-AE patients . Although virulent or resistant pathogens  were more frequently detected in the C-AE patients to detect more, results did not reach statistical significance . So this classification is less useful on the hospital setting leading that almost the exacerbations been classified as complex and therefore to be treated potentially with anti-pseudomonas antibiotics.On the other hand, we believe that the weight of the variables employed to classify the exacerbations is not the same for each variable. Since low FEV1, to employ oral steroids or having more than ≥4 exacerbations in the last year, have been associated more frequently with Pseudomonas aeruginosa infection -20, we dOur study has some limitations that we would like to highlight. First, the retrospective nature of the study: this could lead to an information bias since some confounding factors could be unequally distributed among groups. Second, sputum culture: although it is recommended to all suspected infectious COPD exacerbations, only a low percentage of patients had it; however, this study is reflecting a real life approach when managing hospitalized COPD exacerbations. Third, the population selected is Spanish: we do not know if these CTS recommendations could influence the outcome in the specific Canadian population with a different health system. The CTS classification of AECOPD, according to the presence of risk factors, was not associated with worse short-term clinical outcomes compared to patients without risk factors; however; this classification is a good tool to detect patients with a worse long-term prognosis.